FIGURE 6.

Determination of the rate constant for the reaction of LPO compound II with hydrogen peroxide to form compound III. LPO (2 μm) was preincubated with urate (400 μm) and then allowed to react with various concentrations of hydrogen peroxide (200–800 μm) at 25 °C in 50 mm phosphate buffer, pH 7.0. A, representative spectral changes followed between 5 ms (thick black line) and 80 s (gray line) using 200 μm H₂O₂. Inset shows the α and β bands in greater detail. B, the formation of compound III was monitored at 593 nm (gray dots), and the data were fitted by single exponential functions (black line); representative data were obtained using 200 μm H₂O₂. C, the averaged observed rate constants k_obs were plotted against the concentration of hydrogen peroxide (black dots). Data are means and standard deviations of triplicate measurements. The slope of the linear fit corresponds to the second-order rate constant of the reaction of LPO compound II with hydrogen peroxide to form compound III.